PROJECT SUMMARY Herpes simplex virus 2 (HSV-2) is a mucosally-transmitted infection for which there is no effective vaccine. Human studies of latent HSV-2 infection strongly suggest that resident-memory CD8+ T cells (CD8 TRM) may protect against viral reactivation, but it remains unclear whether CD8 TRM have the capacity to provide sterilizing mucosal immunity against primary infection. Here, I propose to use a novel mouse model of vaccine- induced vaginal CD8 TRM to investigate whether a solely CD8+ T cell-based response can protect against vaginal HSV-2 challenge. In this model, mice are immunized with Listeria monocytogenes engineered to express an immunodominant HSV-derived T cell epitope (LM-gB). Preliminary investigations revealed that mice immunized with LM-gB generated a population of HSV-specific CD8 TRM that remained detectable in the vagina for at least five months after vaccination. However, while circulating memory cells remained stable in this model, vaginal CD8 TRM underwent a tenfold decay in the months following immunization. Mice with fewer vaginal CD8 TRM responded poorly to HSV-2 challenge, exhibiting delayed T cell expansion and failure to achieve protective immunity. By contrast, mice challenged earlier after immunization, when baseline numbers of vaginal CD8 TRM were substantially higher, responded rapidly to infection and exhibited superior protection against severe disease. Therefore, I propose to use this model of waning vaginal-resident immunity to elucidate the relationship between CD8 TRM number and response kinetics after HSV-2 challenge, and establish whether CD8 TRM induced by parenteral vaccination can mediate protective immunity. Finally, I will determine whether mucosal LM-gB immunization enhances CD8 TRM lodgement and protective function. Together, the aims of this proposal will clarify the role of mucosal CD8 TRM in genital HSV-2 infection, and shed light on how to leverage the protective function of this memory population against HSV-2 and other sexually- transmitted viral pathogens.